Proteome analysis of<b><i>Schizosaccharomyces pombe</i></b>by two‐dimensional gel electrophoresis and mass spectrometry

Hwang, Kyu Ri; Carapito, Christine; Böhmer, Susanne; Leize, Emmanuelle; Dorsselaer, Alain Van; Bernhardt, Rita

doi:10.1002/pmic.200500863

Cited by 19 publications

(21 citation statements)

References 46 publications

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“…We were able to identify a large number of proteins (950 representing 549 different proteins), which have been classified according to their function with a distribution similar to that reported for the ascomycete S. pombe (18). Because P. chrysogenum is important for the production of secondary metabolites, especially penicillins, we focused on the proteins related to the biosynthesis of these compounds.…”

Section: Discussionmentioning

confidence: 86%

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Proteome Analysis of the Penicillin Producer Penicillium chrysogenum

Jami

Barreiro

Garcı́a-Estrada

et al. 2010

Molecular & Cellular Proteomics

119

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Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by "blue silver" colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin biosynthesis in the high producer strains. These results provide useful information to improve the production of many other bioactive secondary metabolites. Molecular & Cellular Proteomics 9:1182-1198, 2010.

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Section: Discussionmentioning

confidence: 86%

“…This technique has been successfully applied to other ascomycete fungi, such as Schizosaccharomyces pombe (18), some Aspergillus species (19 -21), and Saccharomyces cerevisiae (22,23). Kiel et al (24) have performed a proteomics-based inventory of the proteins present in the microbody matrix of P. chrysogenum.…”

mentioning

confidence: 99%

Proteome Analysis of the Penicillin Producer Penicillium chrysogenum

Jami

Barreiro

Garcı́a-Estrada

et al. 2010

Molecular & Cellular Proteomics

119

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“…The information collected from the yeast experiments complement a proteomic study on the fission yeast previously reported by us [1]. This study provided the identification of 157 proteins after 2-DE of a cell lysate and, therefore, lacked information about proteins with basic pI (e.g.…”

Section: Discussionmentioning

confidence: 99%

Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two‐dimensional reversed‐phase×ion‐pair reversed‐phase HPLC setup

Wörner

Melchior

Delmotte

et al. 2009

J of Separation Science

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A RPxIP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis. Of the 588 proteins identified from two donors, 24% are involved in cholesterol homeostasis or xenobiotic/lipid metabolism. Up to 50% of the identified proteins belong to the group of membrane proteins, difficult to investigate using gel-based proteomic approaches. We further demonstrated the reproducibility and comparability of the approach and reduced the amount of sample load by almost 70% with only minor loss of information about the proteins identified in the samples. The presented study clearly demonstrates the good applicability of the experimental setup to the analysis of subcellular proteomes including large membrane fractions, where only low amounts of sample material are available.

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“…As Sch. pombe also contains many genes and regulatory aspects with similarity to mammals, further proteome analysis of this organism might contribute to the functional characterization of basic cellular mechanisms of eukaryotes (Hwang et al, 2006 ).…”

Section: Proteome Maps Of Yeastsmentioning

confidence: 99%